Method for introducing a polynucleotide into non-adhesively growing plant cells

ABSTRACT

The present invention relates to a method for introducing a polynucleotide into non-adhesively growing plant cells, comprising the following steps: providing a solid support having immobilized thereto the polynucleotide in dry state; contacting the plant cells with the polynucleotide on the solid support so as to obtain transformed plant cells; and optionally washing the plant cells.

PRIORITY

This application corresponds to the national phase of InternationalApplication No. PCT/EP2011/070602 filed Nov. 21, 2011, which, in turn,claims priority to European Patent Application No. 10.191985.0 filedNov. 19, 2010, the contents of which are incorporated by referenceherein in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing that has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 18, 2013, isnamed LNK_133_Sequence_Listing_US_ST25.txt and is 64,154 bytes in size.

BACKGROUND

Plant as well as bacterial or fungal protoplasts are cells in which thecell wall was partially or completely removed by either mechanical orenzymatic treatment. Since 1961, when enzymatic methods of protoplastisolation from plant tissues were reported, these cell-wall-less cellsfaced various periods of “popularity”. In 1970s totipotency of plantprotoplasts was demonstrated by generating fertile plants from thesecells. This led to a “golden age” period during which major methods andtechniques for cell preparation, handling and treatments includingapproaches for DNA uptake were developed. Further expectations wereraised with respect to generation of plants with novel properties usinggenetic manipulation of protoplasts (e.g. nuclear and organelletransformation, or generation of hybrids and cybrids). Protoplasts wereused as a versatile system to study plant cell development andphysiology, cytodifferentiation, organellogenesis, membrane transportand plant virus function and interaction of viruses with plant cells.Recent advances in genomics, transcriptomics, proteomics and discoveryof fluorescent proteins led to “renaissance” of protoplasts in modernscience. Despite regular exploitation of protoplasts to study gene andprotein function application of protoplasts in high-throughput assaysare rather rare. Reasons for this are absence of efficient, practicaland economical methods to handle and maintain cell cultures at largescales. Protoplast isolation is now routine from a wide range of plantspecies. Typically, a protoplast isolation procedure consists of afiltration step to remove large debris after cell wall digestion and oneor several centrifugation steps using solutions osmotically andionically adjusted for a given species to further purify intact and, inspecial cases, specific cell types, e.g. guard cells, epidermis cellsand other cells. Numerous factors, such as different plant material,pre-isolation, isolation and post-isolation physical and chemicalrequirements and nutrient composition of media used and combination ofgrowth regulators, influence division frequencies of protoplasts andsubsequent development of protoplast-derived colonies. Seasonal andinternal clock conditions may influence cell behaviour even in vitro,but very likely is species specific.

After preparation cells are typically used for analysis and subsequentculture immediately. Protoplasts can be used, for example, for drugassays, transient and/or stable transformation or somatic hybridisation.However, as freshly prepared plant protoplasts do not easily take upforeign nucleic acids protocols need to be developed for efficient DNAuptake at large scale.

BART et al: PLANT METHODS, vol. 2, page 13 (2006) describes a novelsystem for gene silencing using siRNAs in rice leaf and stem-derivedprotoplasts. The protoplasts were transformed with various plasmidsusing PEG as a transformation agent. For the transformation the DNA wasdissolved in a liquid.

YAMADA et al: METHODS IN MOLECULAR BIOLOGY, vol. 643 (2010), pages 33-45describes protocols for the identification of regulatory protein genesinvolved in alkaloid biosynthesis using a transient RNAi system.Transformation is carried out using PEG as transformation agent and DNAdissolved in a liquid.

CRAIG et al: PLANT CELL REPORTS, vol. 24, no. 10 (2005), pages 603-611compares particle bombardment of leaf explants and PEG-mediatedtransformation of protoplasts. The nucleic acids used for PEG-mediatedtransformation were dissolved in a liquid.

YOO et al: NATURE PROTOCOLS vol. 2, no. 7 (2007), pages 1565-1572investigates Arabidopsis mesophyll protoplasts as a cell system fortransient gene expression analysis. “DNA-PEG-calcium transfection” usingDNA in solution is described.

An Advertising Feature of GenVault Corporation, Carlsbad, Calif., USA[Kansagara et al: NATURE METHODS, vol. 5 (September 2008)] describesdry-state, room-temperature storage of DNA and RNA. The nucleic acidsstored in this way, however, cannot be directly used in their dry state.Rather, they first have to be eluted and purified before further use,e.g. in transformation.

In contrast to many human and animal cell cultures (e.g. fibroblasts,pancreatic islet cells, human colon cancer cells and many others) plantprotoplasts are an example of non-adhesively growing cells. So far onlyliquid cultured protoplasts could be used in assays enablinghigh-throughput analysis. There are several drawbacks of liquid culture.The main one is the impossibility to find the same object/cell formicroscopy observation again and again over continuous time periodswhenever container with cultured protoplasts should be translocated ormoved. This becomes particularly essential if e.g. multiple emissionchannels are to be compared and analyzed by means of computationaltools. Only a switch between 2 channels may lead to microvibrationsresulting in cell translocation and thus in a shift between differentchannels. Another limitation is not-avoidable cell aggregation whencultured in liquid medium over continuous time period. This makesimpossible appropriate analysis by e.g. microscopy means. In addition,protoplast populations often consist of more than one cell type byorigin, which could additionally be at different developmental states.The cellular heterogeneity and data extrapolation is a problem, andliquid culture does not allow to solve it.

Immobilisation of non-adhesively growing cells is necessary to preventnon-predictable and uncontrolled cell movement, which is not avoidableif cells float freely in the culture medium. Protoplast embedding intosemi-solid matrixes allows developing cells to generatemicroenvironments. Numerous reports demonstrated that immobilisation ofplant protoplasts resulted in higher plating efficiencies and optimisedcell development. Furthermore, effect of drugs and/or physiologicallyactive compounds can be easily investigated by replacing ofincubation/culture media. Immobilised cells or surface growing cellscould be subjected to automated microscopy to generate image datasuitable for statistical analysis afterwards.

US 2002/173037 A1 describes a method of protoplast culture whichcomprises mixing protoplasts with alginate solution, placing a CaCl₂solution on a glass microslide, placing a mixture of protoplasts andalginate solution on the glass microslide and immediately covering by aglass coverglass, adding CaCl₂ solution in an amount of 70 to 100 μlfrom the sides of coverglass, sliding down the coverglass towards oneside after four to ten minutes and placing it in a petridish containingprotoplast culture medium, sealing the petridishes with parafilm andincubating in dark/diffused light at 20 to 27° C., and transferring theextra thin alginate layer with 20-25 celled colonies to regenerationmedium for development of culture. This process is rather cumbersome,e.g. the coverglasses have to be handled by forceps (see FIG. 1). Thus,it is not suitable for a high-throughput screening or a fully automatedprocess.

Golds et al: J PLANT PHYSIOL, vol. 140, pages 582-587 established the“thin alginate layer” (TAL) technique, in which protoplasts are enmeshedin an alginate medium and placed in liquid culture medium.

PATI al.: PROTOPLASMA, vol. 226, no. 3-4 (2005), pages 217-221 developed“extra thin alginate films” (ETAF) in order to establish a technique forprotoplast culture. The ETAF technique described in this referencerequires placing protoplasts on a microscope slide and placing acoverglass on top of the cells. The coverglass is then removed with thehelp of jeweler's forceps. This technique is not suitable for ahigh-throughput screening or a fully automated process due to the rathercomplicated handling involving coverglasses and forceps.

The TAL technique may be suitable for cell tracking, but this willrequire transfer of the carrier into a plate/container appropriate formicroscopy. In addition, this method cannot be used for automation ofhandling procedures and is based on exclusively man-operatedmanipulation. Also, the TAL-technique is not suitable forhigh-throughput analysis since culture of polypropylene grids takesplace in liquid environment, in which carriers are swimming, rotatingetc. and not in multiwell format. This causes movement of the carrierwith embedded protoplasts and without manual adjustments it isimpossible to find the same object of interest again. Further, thismethod is not suitable for multi-well format.

The same criteria apply to the ETAF technique. This method isexclusively man-powered, handling is complicated and not suitable forhigh-throughput analysis. In addition, non-skilled persons cannot avoidhigh rates of contamination during manipulation. It requirestranslocation of the formed film by manual manipulations and cannot beautomated, thus omitting high-throughput-oriented assays.

Despite existing procedures to immobilise non-adhesively growing cellssuch as plant protoplasts, none of them is suited efficiently for both,high-throughput and high-content analysis in combination with highresolution microscopy analysis, such as TIRF (Total Internal ReflectionFluorescence) microscopy. Established procedures result in cell trappingat various focal planes, thus increasing impact of artefacts on dataquality while performing image analysis. The present invention providesmethods which can be carried out in an automated manner, e.g. inhigh-throughput analysis, and thus allows successfully to overcome mostof the above-mentioned obstacles.

The present invention provides an efficient method for high-throughputsingle cell analysis using robotic handling and automated microscopy. Itwas surprisingly found that the use of dried DNA for transforming plantcells resulted in highly reproducible transformation efficiencies whichis important for automation of the transformation process. Especiallythe variation in the co-transformation efficiency was lower as comparedto known transformation techniques using DNA dissolved in a liquid (seeExample 5). It was further found that the transformation efficiencyusing the dried DNA was very low unless the cells were sedimented priorto or during the transformation.

SUMMARY OF THE INVENTION

A first aspect of the present invention is a method for introducing apolynucleotide into plant protoplast cells, comprising the followingsteps:

-   -   (a) providing a solid support having immobilized thereto the        polynucleotide in dry state;    -   (b) contacting the plant protoplasts with the polynucleotide on        the solid support so as to obtain transformed plant protoplast        cells; and    -   (c) optionally washing the plant protoplasts.

Another aspect of this invention is an automated method for analyzingcells, comprising the following steps:

-   -   providing a culture of non-adhesively growing cells, preferably        of plant protoplast cells;    -   arranging the cells in a monolayer and immobilizing them in the        monolayer; and    -   detecting at least one parameter by microscopic analysis.

The non-adhesively growing cells according to this aspect include animalcells, yeast cells and plant cells. Preferably, the non-adhesivelygrowing cells are plant protoplast cells.

Yet another aspect of the invention is a screening method to identifyefficient artificial microRNA sequences, comprising the following steps:

-   -   introducing a plasmid into plant protoplast cells so as to        obtain transformed plant protoplast cells, wherein said plasmid        comprises a nucleic acid sequence encoding a candidate        artificial microRNA, a nucleic acid sequence representing the        target gene of the candidate artificial microRNA, and optionally        a nucleic acid sequence encoding a transformation marker;    -   culturing the transformed plant protoplast cells under        conditions that allow expression at least of the nucleic acid        sequence encoding the candidate artificial microRNA, and of the        nucleic acid sequence representing the target gene of the        candidate artificial microRNA;    -   selecting as efficient microRNA that candidate artificial        microRNA sequence which is capable of efficiently inhibiting        expression of the target gene.

Accordingly, the present invention relates to the following embodiments:

(1) A method for introducing a polynucleotide into non-adhesivelygrowing eukaryotic cells from either plant or animal origin, comprisingthe following steps:

-   -   (a) providing a solid support having immobilized thereto the        polynucleotide in dry state;    -   (b) contacting the non-adhesively growing cells with the        polynucleotide on the solid support so as to obtain transformed        non-adhesively growing cells, wherein step (b) comprises        -   (i) adding to the solid support a suspension comprising the            non-adhesively growing cells,        -   (ii) arranging the non-adhesively growing cells in a layer            on the solid support,        -   (iii) adding a transformation agent to the suspension; and        -   (iv) optionally removing the transformation agent from the            non-adhesively growing cells;    -   and    -   (c) optionally washing the non-adhesively growing cells.

(2) The method of item (1), wherein step (a) comprises adding a solutioncontaining the polynucleotide onto the solid support and removing thewater from the solution on the solid support.

(3) The method of item (1) or (2), wherein after step (iii) thenon-adhesively growing cells are incubated for 1 to 30 minutes in thepresence of the transformation agent so as to obtain the transformednon-adhesively growing cells.

(4) The method of any one of items (1) to (3), wherein saidtransformation agent is selected from the group consisting ofpolyethylene glycol (PEG), poly-L-ornithine, polyvinyl alcohol anddivalent ions.

(5) The method according to any one of items (1) to (4), wherein atleast 3 different polynucleotides are immobilized on the same solidsupport, each polynucleotide being spatially separated from the otherpolynucleotides.

(6) The method according to item (5), wherein said solid support has aplurality of locations, preferably cavities, and each polynucleotide isimmobilized at a separate location, preferably at the bottom of aseparate cavity.

(7) The method of any one of items (1) to (6), wherein saidnon-adhesively growing cells are plant protoplast cells.

(8) The method of any one of items (1) to (7), wherein saidpolynucleotide comprises a nucleic acid sequence encoding artificialmicroRNA, a nucleic acid sequence representing the target gene of theartificial microRNA, and optionally a nucleic acid sequence encoding atransformation marker.

(9) A method for analyzing non-adhesively growing cells, comprising thefollowing steps:

introducing a polynucleotide into non-adhesively growing cells by amethod according to any one of items (1) to (8) to obtain transformednon-adhesively growing cells;

culturing the transformed non-adhesively growing cells under conditionsthat allow expression of at least one coding sequence comprised in thepolynucleotide;

arranging the transformed non-adhesively growing cells in a monolayerand immobilizing them in the monolayer; and

detecting at least one parameter by microscopic analysis.

(10) The method of item (9), wherein the immobilization of thenon-adhesively growing cells in a monolayer is achieved by adding agelling substance to the non-adhesively growing cells, centrifuging theprotoplast cells to obtain a monolayer of non-adhesively growing cells,and solidifying the gelling substance to form a gel in which thenon-adhesively growing cells are embedded.

(11) The method of item (9) or (10), wherein said at least one parameteris selected from the group consisting of fluorescence, luminescence,morphology and combinations thereof.

(12) A screening method to identify efficient plant microRNA sequences,comprising the following steps:

introducing a polynucleotide into non-adhesively growing cells by themethod of item (8) so as to obtain transformed plant protoplast cells;

culturing the transformed non-adhesively growing cells under conditionsthat allow expression at least of the nucleic acid sequence encoding thecandidate artificial microRNA, and of the nucleic acid sequencerepresenting the target gene of the candidate artificial microRNA;

selecting as efficient microRNA that candidate artificial microRNAsequence which is capable of inhibiting expression of the target gene.

(13) The screening method of item (12), wherein the target gene islabeled with a first fluorescent protein, and the transformation markeris a second fluorescent protein.

(14) The screening method of item (10) or (11), wherein at least 24different artificial microRNAs are examined in one screening cycle usingone single solid support.

(15) The screening method of any one of items (12) to (14), wherein theinhibition of expression of the target gene is determined by microscopy.

(16) An automated method for analyzing a cell, comprising the followingsteps:

-   -   providing a culture of non-adhesively growing cells;    -   arranging the non-adhesively growing cells in a monolayer and        immobilizing them in the monolayer; and    -   detecting at least one parameter by microscopic analysis.

(17) The method of item (16), wherein said non-adhesively growing cellsare plant protoplast cells.

(18) The method of item (16) or (17), wherein the immobilization of thenon-adhesively growing cells in a monolayer is achieved by adding agelling substance to the non-adhesively growing cells, centrifuging theprotoplast cells to obtain a monolayer of non-adhesively growing cells,and solidifying the gelling substance to form a gel in which thenon-adhesively growing cells are embedded.

(19) The method of any one of items (16) or (18), wherein said at leastone parameter is selected from the group consisting of fluorescence,luminescence, morphology and combinations thereof.

(20) The method of any one of items (16) or (19), wherein saidnon-adhesively growing cells have been transformed with a polynucleotideto arranging them in a monolayer, preferably by a method as defined inany one of items (1) to (8).

(21) The method of item (20), wherein said polynucleotide is a lineardouble-stranded DNA consisting of a promoter, an open reading frame, anda terminator, and wherein said nucleic acid is directly used for thetransformation without inserting it into a plasmid.

(22) A screening method to identify efficient artificial microRNAsequences, comprising the following steps:

-   -   introducing a plasmid into non-adhesively growing plant cells so        as to obtain transformed plant protoplast cells, wherein said        plasmid comprises a nucleic acid sequence encoding a candidate        artificial microRNA, a nucleic acid sequence representing the        target gene of the candidate artificial microRNA, and optionally        a nucleic acid sequence encoding a transformation marker;    -   culturing the transformed non-adhesively growing plant cells        under conditions that allow expression at least of the nucleic        acid sequence encoding the candidate artificial microRNA, and of        the nucleic acid sequence representing the target gene of the        candidate artificial microRNA;    -   determining the inhibition of expression of the target gene by        the candidate artificial microRNA; and    -   selecting as efficient microRNA that candidate artificial        microRNA sequence which is capable of efficiently inhibiting        expression of the target gene.

(23) The screening method of item (22), wherein the target gene islabeled with a first fluorescent protein, and the transformation markeris a second fluorescent protein.

(24) The screening method of item (22) or (23), wherein at least 24different candidate artificial microRNAs are examined in one screeningcycle.

(25) The screening method of any one of items (22) to (24), wherein theinhibition of expression of the target gene is determined by microscopy.

(26) The screening method of any one of items (22) to (25), wherein theplasmid is transformed into non-adhesively growing plant cells by amethod as defined in any one of items (1) to (8).

(27) The screening method of any one of items (22) to (25), wherein,prior to the step of determining the inhibition of expression of thetarget gene, the transformed non-adhesively growing plant cells arearranged in a monolayer and immobilized in the monolayer, preferably asdefined in item (18).

(28) The method of any one of items (1) to (15), wherein saidpolynucleotide is a linear double-stranded DNA consisting of a promoter,an open reading frame, and a terminator, and wherein said nucleic acidis directly used for the transformation without inserting it into aplasmid.

DETAILED DESCRIPTION Definitions

In the context of this disclosure, a number of terms and abbreviationsare used. The following definitions are provided.

“microRNA or miRNA” refers to oligoribonucleic acid, which regulatesexpression of a polynucleotide comprising the target sequence. microRNAs(miRNAs) are noncoding RNAs of about 19 to about 24 nucleotides (nt) inlength that have been identified in both animals and plants whichregulate expression of a polynucleotide comprising the target sequence.They are processed from longer precursor transcripts that range in sizefrom approximately 70 to 2000 nt or longer, and these precursortranscripts have the ability to form stable hairpin structures. Inplants, miRNAs usually have single, highly complementary target sitesthat mostly locate to coding regions. A miRNA is an “artificial miRNA”when it is genetically engineered. The artificial miRNA is thuspredetermined to specifically target a single gene or multiple genes.

“pri-miRNAs” or “primary miRNAs” are long, polyadenylated RNAstranscribed by RNA polymerase II that encode miRNAs. “pre-miRNAs” areprimary miRNAs that have been processed to form a shorter sequence thathas the capacity to form a stable hairpin and is further processed torelease a miRNA.

A “target gene” refers to a gene that encodes a target RNA, i.e., a genefrom which a target RNA is transcribed. The gene may encode mRNA, tRNA,small RNA, etc. A “target sequence” refers to an RNA whose expression isto be modulated, e.g., down-regulated. The target sequence may be aportion of an open reading frame, 5′ or 3′ untranslated region, exon(s),intron(s), flanking region, etc.

A “star sequence” or “miRNA* strand” is the complementary sequencewithin a miRNA precursor that forms a duplex with the miRNA. Thecomplementarity of the star sequence does not need to be perfect.

A new strategy developed using the knowledge on miRNA biology offered bycombination of the advantages of RNAi and T-DNA insertion techniques. Asplant miRNAs tend to show a high degree of sequence complementarity totheir target RNA, several research groups assumed that miRNAs could beused for gene silencing studies. Based on different endogenous miRNAprecursor sequences, they designed strategies to replace the21-nucleotide stretch of the mature miRNA against a 21-nucleotidesequence complementary to a given target gene. By simultaneouslyexchanging the 21 nucleotides of the miRNA* strand, the stem-loopstructure of the precursor was preserved and the processing resulted ina novel miRNA/miRNA* duplex against a chosen target gene. Theiradvantage lies in the specificity of sequence homology, based on theshort length of only 21 nucleotides. They could therefore be applied forthe knock-down of single as well as multiple genes with a singleconstruct.

A recombinant construct comprises an artificial combination of nucleicacid fragments, e.g., regulatory and coding sequences that are not foundtogether in nature. The construct may be transcribed to form an RNA,wherein the RNA may be capable of forming a double-stranded RNA and/orhairpin structure. This construct may be expressed in the cell, orisolated or synthetically produced. The construct may further comprise apromoter, or other sequences which facilitate manipulation or expressionof the construct.

As used here “suppression” or “silencing” or “inhibition” are usedinterchangeably to denote the down-regulation of the expression of aproduct of a target sequence. If the suppression by an artificial miRNAis concerned, the degree of suppression by this artificial miRNA isdetermined relative to the same organism lacking the nucleic acidencoding the artificial miRNA (e.g. relative to a cell comprising thesame target sequence which, however, lacks the nucleic acid sequenceencoding the artificial miRNA). This “same organism” (e.g. a cell)should be identical to the test organism (cell) comprising the nucleicacid encoding the artificial miRNA, except that the nucleic acidsequence encoding the artificial miRNA to be tested is absent.Suppression includes expression that is decreased by at least about 10%,preferably by at least about 25%, more preferably by at least about 50%,more preferably by at least about 75%, most preferably by at least about90%, e.g. by about 95% or about 100% relative to the same organism (e.g.a cell) which lacks the nucleic acid sequence encoding the artificialmicroRNA.

As used herein, “encodes” or “encoding” refers to a DNA sequence whichcan be processed to generate an RNA and/or polypeptide.

As used herein, “expression” or “expressing” refers to production of afunctional product, such as, the generation of an RNA transcript from aDNA sequence. The term may also refer to a polypeptide produced from anmRNA generated from a DNA precursor. Thus, expression of a nucleic acidfragment may refer to transcription of the nucleic acid fragment and/ortranslation of RNA into a polypeptide.

“Plant” includes reference to whole plants, plant organs, plant tissues,seeds and plant cells and progeny of same. Plant cells include, withoutlimitation, cells from seeds, suspension cultures, embryos, meristematicregions, callus tissue, leaves, roots, shoots, gametophytes,sporophytes, pollen, and microspores. Preferably, the plant cells usedherein are monocotyledonous or dicotyledonous plant cells, but alsolower plants such as algae or mosses like Physcomitrella patens or else.

An example of a monocotyledonous cell is a maize cell. Preferably, theplant cell is a dicot plant cell. Examples of dicot plant cells includesoybean, rapeseed, sunflower, flax, cotton, barley, bean, pea, tobacco,and Arabidopsis.

The term “introduced” means providing a nucleic acid (e.g., expressionconstruct) or protein into a cell. Introduction of nucleic acid intoplant cells is referred to herein also as “transformation”. Thetransformation may be transient or stable.

Method for Introducing a Polynucleotide into Plant Protoplast Cells

According to a first aspect, the present invention pertains to a methodfor introducing a polynucleotide into plant protoplast cells, comprisingthe following steps: (a) providing a solid support having immobilizedthereto the polynucleotide in dry state; (b) contacting the plantprotoplasts with the polynucleotide on the solid support so as to obtaintransformed plant protoplast cells; and (c) optionally washing the plantprotoplasts.

Preferably, the plant protoplast cells used herein are derived frommonocotyledonous or dicotyledonous plants or lower plants. The phrase“derived from” means “obtained from” or “isolated from”. An example of amonocotyledonous plant is maize. Preferably, the plant protoplast cellsare derived from dicotyledonous plants. Examples of dicotyledonousplants include soybean, rapeseed, sunflower, flax, cotton, barley, bean,pea, tobacco, and Arabidopsis. Most preferably, the plant protoplastcells are derived from Arabidopsis, e.g. Arabidopsis thaliana.

Methods for isolating plant protoplast cells are known to the skilledperson. Suitable protocols can be found in, e.g., Arabidopsis protocols,2^(nd) edition 2005 (Methods in molecular biology) edited by JulioSalinas and Jose J. Sanchez-Serrano (ISBN 978-1-61737-539-2); Davey andAnthony, Plant Cell Culture: Essential Methods, 1^(st) ed. 2010, (ISBN978-0470686485).

The solid support may be made of any material which does not adverselyaffect the growth of plant protoplast cells. Preferably, the solidsupport does not contain tungsten or gold, or it does not consist oftungsten or gold. More preferably the solid support does not contain ametal, or it does not consist of a metal. The solid support ispreferably made of a water-impermeable material. Suitable materialsinclude, but are not limited to, glass, polystyrene, polypropylene,polycarbonate. Preferably, the solid support is suitable to allow one ormore of optical absorbance, fluorescence and luminescence detection.Typically, the solid support comprises one or more planar or concavesurfaces; and/or the solid support does not have a spherical form. In apreferred embodiment, the solid support has a plurality of locations,preferably cavities, where different polynucleotides may be immobilized.This embodiment is preferably a multi-well plate having a plurality of“wells” or “cavities”. At the bottom of the cavities, the surface ispreferably planar or concave. Suitable types include multi-well cultureplates in 6-, 12-, 24-, 48-, 96-, 384- or higher well formats. Preferredare 24-well plates, more preferred are 48-well plates, most preferredare 96-well plates. The nominal volume of each well is preferably from0.1 ml to about 2 ml, most preferably it is about 0.5-1 ml.

In a preferred embodiment, the polynucleotide is immobilized on thesolid support by adding a solution containing the polynucleotide ontothe solid support and removing the water from the solution on the solidsupport. The removal of the water can be achieved by letting evaporatethe water over about 6-48 h, preferably over about 12-36 h, e.g. 24 h.This is preferably done under sterile conditions, e.g. under a sterileflow hood. Alternatively, the water may be removed by vacuumexsiccation.

Typically, 0.1 μg to 10 μg, preferably 0.2 μg to 5 μg, more preferably0.3 μg to 2 μg, most preferably 0.5 μg to 1.5 μg of (each)polynucleotide is added to the solid support or to each separatelocation of the solid support.

The dried DNA immobilized on the solid support can be stored, e.g. at−20° C. or lower, for at least 1 month, e.g. for at least 2 or 3 or 4 or5 or 6 months or 12 months or longer.

The transformation step may comprise adding to the solid support asuspension comprising the plant protoplasts. The protoplasts arepreferably suspended in a suitable medium that does not adversely affector inhibit later transformation, e.g. TM550 (see Table 1 infra). As anexample, the following medium can be used: 0.5 mM MES (salt-free), 15 mMMgCl₂, 0.48 mM mannitol, pH 5.8 (TM550).

The cell density in the protoplast suspension may range from about 1×10⁴to about 1×10⁸, preferably from about 1×10⁵ to about 1×10⁷, morepreferably from about 5×10⁵ to about 2×10⁵, most preferably it is about1×10⁶ protoplast cells per ml. The suspension of non-adhesively growingplant cells may be added directly to the dried DNA on the solid support.Alternatively, the dried DNA may first be re-dissolved in a suitablesolution, followed by addition of the cell suspension. Preferably, 10 μlto 500 μl, more preferably 20 μl to 200 μl, still more preferably 25 μlto 100 μl, most preferably 30 μl to 50 μl of protoplast suspension isadded to the DNA, e.g. in a cavity of a multi-well plate.

Afterwards the cells are arranged in a layer on the solid support.Typically, the cells are sedimented so as to arrange them in a layer onthe solid support (e.g. at the bottom of a well or cavity of amulti-well plate). The term “sedimenting”, as used herein, includesactively sedimenting the cells by applying a centrifugal force to thecells, and passively sedimenting the cells, i.e. allowing the cells tosediment (settle) on the solid support by way of the normal gravity. Thecells may be allowed to sediment for about 0.3 to 60 min, preferably forabout 0.5 to 10 min, most preferably for about 1 to 2 minutes (passivesedimentation). Alternatively, protoplasts could be centrifuged for atleast 30 seconds at least 2 g, e.g. for 1 min at 10 g (Activesedimentation). After the sedimentation step the cells are arranged in alayer, preferably a monolayer, on the solid support, e.g. at the bottomof a well or cavity of a multi-well plate.

Next the transformation of the cells is effected, preferably by achemically induced nucleic acid uptake. Suitable procedures are known tothose of skill in the art (Negrutiu I, Shillito R, Potrykus I, BiasiniG, Sala F (1987) Hybrid genes in the analysis of transformationconditions. I. Setting up a simple method for direct gene transfer inplant protoplasts. Plant Mol Biol 8: 363-373; Koop H U, Steinmuller K,Wagner H, Rössler C, Eibl C, Sacher L (1996) Integration of foreignsequences into the tobacco plastome via polyethylene glycol-mediatedprotoplast transformation. Planta, 199:193-201; Yoo S D, Cho Y H, SheenJ (2007) Arabidopsis mesophyll protoplasts: a versatile cell system fortransient gene expression analysis. Nat Protoc 2:1565-1572). Preferably,a transformation agent is added to the protoplast suspension comprisingthe polynucleotide in order to induce nucleic acid uptake. Thetransformation agent may be polyethylene glycol (PEG) or anothersuitable agent which induces DNA uptake into protoplasts. Alternativetransformation agents include poly-L-ornithine, polyvinyl alcohol anddivalent ions. Preferably, the PEG is PEG 1500. The transformation agentis usually comprised in a solution which is added to protoplastsuspension. For example, an equal volume of 40% PEG 1500 may be added tothe protoplast suspension. A preferred composition to be added to theprotoplast suspension is as follows: 67 mM Ca(NO₃)₂.4H₂O, 270 mMMannitol, 384 g/l PEG1500, pH 9.75 (see also Table 1 infra).

After addition, the suspension is preferably incubated for about 7 to 10minutes. After that, TM550 may be added, preferably about 40 to 60% ofthe volume of the suspension (protoplasts+transformation agentcomposition) already present in the well. After about further 1 to 3minutes, e.g. 2 minutes, a suitable solution (e.g. TM550) is added toincrease the total volume to about 1 ml. The protoplasts may be washedonce or several times with a suitable medium, e.g. TM550, in order toremove the transformation agent, e.g. PEG, and Ca²⁺ ions. After thewashing, the transformed protoplasts may be resuspended in a suitablesolution, e.g. PCA (see Table 2 infra).

As mentioned supra, the solid support is preferably a multi-well plate.Accordingly, it is preferred that a plurality of differentpolynucleotides are immobilized in different wells of the plate,respectively. Preferably, the number of different polynucleotides on thesame solid support is at least 2, more preferably at least 6, morepreferably at least 12, most preferably at least 24, e.g. 48 or 96. Itis important that each polynucleotide is spatially separated from theother polynucleotides. This is of course accomplished if eachpolynucleotide is immobilized at the bottom of a different well of amulti-well plate.

The polynucleotide is preferably plasmid DNA. The polynucleotide maycomprise various nucleic acid sequences encoding different products.Usually, the polynucleotide comprises a nucleic acid sequence encoding atransformation marker. Suitable transformation markers includefluorescent proteins, e.g. red fluorescent protein (“mCherry”) or greenfluorescent protein (GFP). In another embodiment, the polynucleotidecomprises a nucleic acid sequence encoding plant microRNA. In apreferred embodiment, the polynucleotide comprises a nucleic acidsequence encoding a plant microRNA, and a nucleic acid sequence encodingthe corresponding target gene. In the most preferred embodiment, thepolynucleotide comprises a nucleic acid sequence encoding an artificialmicroRNA, a nucleic acid sequence representing the target gene of theartificial microRNA, and a nucleic acid sequence encoding atransformation marker. These nucleic acid sequences are preferablypresent on a single DNA plasmid. Preferred embodiments of the nucleicacid sequences, of the vectors and plasmids that may be used aredisclosed infra in respect of the method of screening. These embodimentsapply to this first aspect of the invention mutatis mutandis.

The DNA uptake method of this invention can be carried out in a fullyautomated manner. A particular advantage is that the plates having thedried DNA immobilized thereto can be stored and shipped for later use,without loss in transformation efficiency. Another important advantageis that only small amounts of a plasmid are required for foreignpolynucleotides uptake.

Monolayer Embedding of Non-Adhesively Growing Cells

In another aspect, this invention relates to an automated method foranalyzing cells, comprising the following steps

-   -   providing a culture of non-adhesively growing cells, preferably        of plant protoplast cells;    -   arranging the cells in a monolayer;    -   immobilizing the cells in the monolayer; and    -   detecting at least one parameter by microscopic analysis.

The non-adhesively growing cells according to this aspect include animalcells, yeast cells and plant cells. Preferably, the non-adhesivelygrowing cells are plant protoplast cells.

Protoplasts can be isolated and cultured by known methods, see supra.The protoplast culture may be provided in wells of a multi-well plate asdescribed supra with respect to the transformation method of theinvention. The protoplasts may or may not be transformed. Theprotoplasts are then suspended in a suitable immobilization medium whichcomprises at least one gelling substance. A gelling substance is asubstance that can convert a solution into a gel. The conversion from asolution into a gel may require cooling or addition of divalent metalions such as Ca²⁺. The gelling substance may be a water-solublepolysaccharide. Gelling substances include but are not limited to agar,κ-carrageenan, I-carrageenan, alginic acid, alginate, agarose,furcellaran, jellan gum, glucono-δ-lactone, azotobactor vinelandii gum,xanthan gum, pectin, guar gum, locust bean gum, tara gum, cassia gum,glucomannan, tragacanth gum, karaya gum, pullulan, gum arabic,arabinogalactan, dextran, sodium carboxymethyl cellulose, methylcellulose, cyalume seed gum, starch, chitin, chitosan, and curdlan.Preferred gelling substances according to this invention include but arenot limited to low melting temperature agarose, agar and alginic acid(ratios and concentrations may vary upon species used, but arepreferably Ca²⁺ free).

The immobilization medium preferably contains mannitol and MES(2-[N-morpholino]ethane-sulfonic acid). The concentration of MES mayrange from 1 mM to about 100 mM, preferably from about 5 mM to about 50mM, most preferably from about 10 mM to about 20 mM.

The concentration of mannitol in the immobilization medium may rangefrom 10 mM to 1 M, preferably it is from 100 mM to 500 mM. Theimmobilization medium may further comprise calcium chloride, magnesiumchloride and/or magnesium sulfate at suitable concentrations. Preferredimmobilization media are disclosed in the examples section.

If alginic acid is used as a gelling substance the immobilization mediumshould not comprise Ca²⁺ ions. A suitable immobilization medium foralginic acid mediated embedding is described supra. If low meltingtemperature agarose or agar is used as gelling substance, theimmobilization medium may contain calcium ions, e.g. at a concentrationfrom 1 mM to 1 M preferably from 10 mM to 100 mM.

If agarose is used as a gelling substance, the concentration of agarosein the immobilization medium is preferably from 1% (w/w) to 5% (w/w),preferably it is about 2% (w/w). If low melting temperature agarose isused, it is important to maintain the temperature above 30° C.,preferably above 35° C. in order to avoid generation of agaroseaggregates.

The concentration of alginic acid in the immobilization medium ispreferably from 0.5% (w/v) to 5% (w/v), more preferably from 1.5% (w(v)to 3% (w/v).

Next a gravity force is applied to form a cell monolayer at the bottomof the multi-well slide or plate. This can be achieved either byallowing the cells to sediment (passive sedimentation), e.g. for atleast 5 minutes, or by centrifugation for at least 30 seconds at 2 g ormore, e.g. for 1 min at 10 g (active sedimentation).

The cells are then trapped at the bottom of the solid support(multi-well plate or slide) by solidification of the gelling substance.This may be achieved by lowering the temperature to below 40° C.,preferably to below 35° C., preferably to below 30° C. (in case of agaror agarose), or by adding a solution containing at least 10 mM Ca²⁺salts at the top of the protoplasts mixed with the alginic acidcontaining medium as microdrops 5 μl or smaller) (in case of alginicacid as gelling substance). If alginic acid is used as a a gellingsubstance, the gelling is induced by increasing the calciumconcentration to at least 1 mM, preferably to at least 50 mM, morepreferably to at least 125 mM.

For further culture, a suitable culture medium may be added on top ofthe gels formed, e.g. to the nominal filling volume of the well. Theimmobilized protoplast cells can be further cultured in theirimmobilized state by adding suitable media on top of the solidifiedprotoplast composition. Similarly, the immobilized in that wayprotoplasts are accessible to exposure of any test substances in thisstage, i.e. prior to analysis.

The method according to this aspect of the invention therefore comprisesin a specific embodiment the step of contacting the immobilizedprotoplast cells with a test compound, and determining the effect of thetest compound on the protoplast cells. This is preferably done by (i)determining at least one parameter of the cells in the presence of testcompound, (ii) determining at least one parameter of the cells in theabsence of test compound, and (iii) comparing the parameters determinedin (i) and (ii).

The parameters are usually determined by microscopy and include, but arenot limited to, fluorescence, morphology and combinations thereof.Methods of microscopy include, but are not limited to, those describedin, e.g., Hasek J, Streiblová E. Fluorescence microscopy methods.Methods Mol Biol 53, 391-405 (1996), Ehlert A, Weltmeier F, Wang X,Mayer C S, Smeekens S, Vicente-Carbajosa J, Dröge-Laser W. Two-hybridprotein-protein interaction analysis in Arabidopsis protoplasts:establishment of a heterodimerization map of group C and group S bZIPtranscription factors. Plant J 46, 890-900 (2006), Bücherl C, Aker J, deVries S, Borst J W. Probing protein-protein Interactions with FRET-FLIM.Methods Mol Biol 655, 389-399 (2010).

The method of this aspect of the invention is preferably combined with apreceding transformation of the protoplast cells prior toembedding/immobilization. In that embodiment, the method may compriseexpression of transformed nucleic acid sequence(s) and determining theeffect of that expression on the cells. Preferably, the polynucleotidetransformed is one as defined above in respect of the method forintroducing polynucleotides into plant protoplast cells according to thefirst aspect of the invention.

All steps can be carried out in a fully automated manner, e.g. by usingpipetting and dispersing robots known in the art.

Any preferred embodiments described supra in respect of the method forintroducing polynucleotides into plant protoplast cells can be appliedto this aspect mutatis mutandis.

mlRNA Screening Method

Yet another aspect of the invention is a screening method to identifyefficient plant microRNA sequences, comprising the following steps:

-   -   introducing a plasmid into plant protoplast cells so as to        obtain transformed plant protoplast cells, wherein said plasmid        comprises a nucleic acid sequence encoding a candidate        artificial microRNA, a nucleic acid sequence representing the        target gene of the candidate artificial microRNA, and optionally        a nucleic acid sequence encoding a transformation marker;    -   culturing the transformed plant protoplast cells under        conditions that allow expression at least of the nucleic acid        sequence encoding the candidate artificial microRNA, and of the        nucleic acid sequence representing the target gene of the        candidate artificial microRNA;    -   selecting as efficient microRNA that candidate artificial        microRNA sequence which is capable of efficiently inhibiting        expression of the target gene.

The method is preferably carried out in combination with the method forintroducing polynucleotides into plant protoplast cells describedherein. All embodiments described above apply to this screening methodas well.

The screening method uses a vector comprising a nucleic acid sequenceencoding a candidate artificial microRNA, a nucleic acid sequencerepresenting the target gene of the candidate microRNA, and a nucleicacid sequence encoding a transformation marker. These components arepresent on a single vector. This avoids the need for transformation ofmultiple plasmids or the like. Typical elements of plant transformationplasmids may also be included in the plasmid used in accordance withthis invention.

Preferably, the three main elements of the vector, (i) the nucleic acidsequence encoding the candidate artificial microRNA, (ii) the nucleicacid sequence representing the target gene of the candidate microRNA(“the target sequence”), and (iii) the nucleic acid sequence encodingthe transformation marker are expressed in the protoplast cell.Efficient expression can be achieved by using a suitable promoteroperably linked to the respective nucleic acid sequence. Variouspromoters can be used. The promoter may also contain, if desired, apromoter regulatory region (e.g., one conferring inducible,constitutive, environmentally- or developmentally-regulated, or cell- ortissue-specific/selective expression), a transcription initiation startsite, a ribosome binding site, an RNA processing signal, a transcriptiontermination site, and/or a polyadenylation signal. Constitutive,tissue-preferred or inducible promoters can be employed. Examples ofconstitutive promoters include the cauliflower mosaic virus (CaMV) 35Stranscription initiation region, the 1′- or 2′-promoter derived fromT-DNA of Agrobacterium tumefaciens, the ubiquitin 1 promoter, the Smaspromoter, the Nos promoter, the pEmu promoter, the rubisco promoter, theGRP1-8 promoter and other transcription initiation regions from variousplant genes known to those of skill.

The artificial candidate microRNA may be designed on the basis of knowndesign tools, e.g. that described in Ossowski, S., Schwab, R. & Weigel,D. Gene silencing in plants using artificial microRNAs and other smallRNAs. Plant J 53, 674-690 (2008). However, it surprisingly turned outthat only a small fraction of the so designed microRNAs were efficientin silencing the target gene. Thus, there is a need for a screeningprocess in order to identify microRNA sequences which are actuallyefficient in silencing a given target molecule.

Target sequences may include coding regions and non-coding regions suchas promoters, enhancers, terminators, introns and the like. Preferably,target sequences are located within coding regions.

The transformation marker is used to label cells that have beensuccessfully transformed. Suitable transformation markers used in theinvention include, but are not limited to, any flurorescent proteins,e.g. those described in Shaner, N. C. et al. Improved monomeric red,orange and yellow fluorescent proteins derived from Discosoma sp. redfluorescent protein. Nat Biotechnol 22, 1567-1572 (2004).

The nucleic acid encoding the target sequence is preferably fused to anucleic acid encoding a marker, e.g. a fluorescent protein such as greenfluorescent protein, a luminescent protein such as luciferase, or anenzyme which catalyzes a detectable reaction. This “target marker” canthen be detected as a measure of the level of expression of the targetsequence. If the target gene is an enzyme, it may be possible that theexpression product of the target gene is the target marker itself.

The method thus comprises in a preferred embodiment the steps of:

-   -   determining the expression of the “target marker”,    -   comparing the level of expression of the target marker to that        of a control cell, e.g. a protoplast cell transformed with a        control vector comprising a nucleic acid sequence encoding a        mock artificial miRNA or with a control vector lacking a nucleic        acid sequence encoding the candidate miRNA sequence, and    -   selecting the candidate miRNA sequence as efficient miRNA        sequence if the level of expression of the target marker in the        cell transformed with the vector encoding the candidate miRNA is        significantly lower (e.g. by at least 10%, preferably by at        least 25%, more preferably by at least 50%, most preferably by        at least 75% or at least 90%) than that in the control cell        transformed with the control vector.

The present screening method can be advantageously combined with theother aspects of the invention, namely the method for introducing apolynucleotide into plant protoplast cells, and theimmobilization/embedding technique described supra.

A particular embodiment, which is applicable to all aspects of thepresent invention, includes the use of PCR products for transformation,preferably for transient transformation. In the methods of the inventionthe polynucleotide or DNA to be transformed may therefore be a PCRproduct comprising the DNA of interest, preferably a promoter, an openreading frame, and a terminator. The PCR product is directly used fortransformation without cloning it into a plasmid or vector. Mostpreferably, the polynucleotide or DNA to be transformed consists of apromoter, an open reading frame, and a terminator. That is, the PCRproduct contains substantially no flanking regions. The advantage isthat such PCR product will lead to much higher transformationefficiencies than PCR products including flanking regions (see Example9).

The use of PCR products for transformation is advantageous as it is muchfaster than the classical cloning approach involving the use of plasmidsfor transformation. This embodiment is therefore particularly suited forautomated methods and high-throughput processes.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Illustration of main steps for transient transformation of cellsin microwells using dried nucleic acids (A) and for cell immobilisationbased on cell sedimentation and subsequent trapping at the bottom ofmicrowells either in alginic acid medium (B) or low melting temperaturegelling substances (C).

FIG. 2. Alginate matrix formation after adding microdroplets of W5.

FIG. 3. Development of tobacco leaf protoplasts using PME. Bright field(A, C, E, G, I, K) and chlorophyll autofluorescence channel (B, D, F, H,J, L) images were acquired with 24 h interval after embedding (A, B).Scale bar=60 μm.

FIG. 4. Effect of compound R113 do we need to disclose this compound asthis will be subject of another patent application to be filed soon,Alternative: kinase inhibitor? on tobacco leaf protoplast developmentafter 6 days of culture. Normal development of protoplasts cultured inF-PCN medium (A) or in F-PCN with 1 μm of R113 (B). Inhibition of celldivisions and colony formation in F-PCN medium supplemented with 10 μMof R113 (C), complete inhibition of cell division and cell death inF-PCN supplemented with 25 μM of R113 (D). Scale bar=60 μm.

FIG. 5. Tracking of developing protoplasts from Arabidopsis seedlings.Images were acquired after 24 h (A), 48 h (B) and 96 h of culture usingthe PME-technique. Scale bar=40 μm.

FIG. 6. Immobilization of protoplasts from DR5-GFP Arabidopsis markerline. Bright field (A,C) and GFP channel (B,D) images were acquiredright after immobilization (A,B) and after 72 h of culture (C,D)demonstrating an increase in promoter activity in cells at later timepoint. Scale bar=40 μm.

FIG. 7. Efficiency of transient transformation of Arabidopsis leafprotoplasts using dried DNA (FIG. 14, Table 3). Expression of mCherryprotein driven under control of the rolD promoter was estimated after 24h. Data represents mean values of three independent experiments, errorbars indicate standard deviation.

FIG. 8. Co-transformation of Arabidopsis protoplasts using dried anddissolved DNA.

FIG. 9. Transient transformation of tobacco leaf protoplasts using driedDNA. Effect of transient expression of 35S::PIN8-Venus (FIG. 15)construct on protoplast development (A-E). Arrow heads in bright fieldimages (A, C, E) mark positions of transiently transformed cellsidentified in YFP channel (B, D) after 24 h (A,B), 96 n (C,D) and 144 h(E) of culture. Scale bar=60 μm. Efficiency of transient transformationof tobacco protoplasts using dried plasmid DNA: 1-1 μg of 35S::Venus,2-2 μg of 35S::Venus (FIG. 16), 3-1 μg of 35S::ER-YFP, 4-2 μg of35S::ER-YFP, 5-2 μg of 35S::Golgi-YFP, 6-2 μg of 35S::PIN1-Venus, 7-1 μgof 35S::PIN8-Venus, 8-2 μg of 35S::PIN8-Venus. All constructs except ofPIN1 and PIN8 tagged with yellow fluorescent protein Venus were takenfrom Nelson et al. A multicoloured set of in vivo organelle markers forco-localization studies in Arabidopsis and other plants. Plant J 51,1126-1136 (2006). Data represents mean values of three independentexperiments, error bars indicate standard deviation.

FIG. 10. Tracking and 3D reconstructions of developing tobaccoprotoplasts after the PME immobilization. Image were acquired with 24 hinterval starting 24 h after PEG mediated DNA uptake. Differentsubcellular compartments where visualised using subcellular markerstranslationaly fused to fluorescent proteins: cytoplasmic YFP (A),mitochondrial YFP (B), endoplasmatic reticulum mCherry (C) and tonoplastmCherry (D). All constructs were taken from Nelson et al. Amulticoloured set of in vivo organelle markers for co-localizationstudies in Arabidopsis and other plants. Plant J 51, 1126-1136 (2006).Scale bar=40 μm

FIG. 11. Knock-down of a stable reporter gene (GFP) by artificialmicroRNAs (FIG. 14, Table 3). Mean absolute GFP pixel intensity oftransformed cells over time (57±20 cells per sample per day).

FIG. 12. Validation of artificial microRNAs efficiency by knock-down ofthe transiently expressed target gene, PIN1, translationally fused withthe GFP (FIG. 14, Table 3). Transformed cells express the markermCherry, PIN1::mGFP5 and an amiRNA; wt represents cells expressing onlymCherry. Images were taken 2 days after transformation, asterisksindicate significant (*, p<0.05) or highly significant (**, p<0.0005)difference to miR³¹⁹a sample; bars represent median pixel intensityvalues of the respective cell population; error bars correspond tostandard error.

FIG. 13. Expression of amiR^(PIN1) in stable transgenic lines (Col-0wild-type background). A: Northern Blot analysis shows expression andprocessing of amiRNAs in several independent lines (upper panel). 5SrRNA serves as loading control (lower panel). DNA oligonucleotidesantisense to the respective amiRNA and end-labelled with γ-ATP were usedas probes. B-C: Representative phenotypes observed among independentlines transformed with the same construct. B: Altered phyllotaxispattern of rosette leaves (arrows) in plants expressing amiR^(P1),amiR^(P2), amiR^(P3) and amiR^(P33) 2 weeks after germination; scalebar=10 mm. C: Altered shoot phyllotaxis and pint-like shoot phenotypes(arrows) in lines expressing amiR^(P1), amiR^(P2) and amiR^(P33) (scalebar=10 mm).

FIG. 14. Map of amiRNA screening vector pMIR-AT1G73590-mGFP5-4consisting of three screening elements under corresponding regulatoryelements: the amiRNA gene (amiRNA and passenger amiRNA* sequences couldbe substituted according to Table 3), the target gene PIN1 fused to thereporter marker GFP (this expression element was present for the example8, but not for the example 7) and the transformation marker mCherry. Thenucleotide sequence of pMIR-AT1G73590-mGFP5-4 is shown in SEQ ID NO:1.

FIG. 15. Map and sequence of the vector p2GW7,0-PIN8-Venus forexpression of the PIN8-Venus construct. The nucleotide sequence ofp2GW7,0-PIN8-Venus is shown in SEQ ID NO:2.

FIG. 16. Map of the vector pAM-PAT-AtPIN1-Venus for expression of thePIN1-Venus construct. The nucleotide sequence of pAM-PAT-AtPIN1-Venus isshown in SEQ ID NO:3.

FIG. 17. A scheme representing primer design for the PPTT experimentshown in Example 9.

FIG. 18. Fluorescence microscopy analysis of transformation efficienciesusing plasmid and PCR-expression cassettes in aqueous solution, 24 hafter transformation (Example 9).

FIG. 19. Concentration-dependent effect of auxin NAA on firefly/renillaratio (Example 9)

FIG. 20. Co-transformation (a) and single-product (b) PCR-based amiRNAscreening strategies. All steps are applicable for automation.

FIG. 21. Effect of PIN5 and PIN8 expression on tobacco leaf protoplastdevelopment. Transient PIN5 expression does not affect developmentalprogram of tobacco leaf protoplasts, while expression of PIN8 arrestscell division and enhances elongation. Arrowheads indicate transformedcells. Scale bars are 20 μm.

FIG. 22. Intracellular localization of AtPIN8-Venus in tobacco leafprotoplasts. Spinning-disk imaging at the cell periphery (a) and insidethe cell (b) as well TIRF imaging near the plasma membrane (c) proves ERlocalization of AtPIN8-Venus (Andromeda microscope, TILL Photonics GmbH,Germany). Scale bars are 5 μm.

FIG. 23. Quantitative analysis of cell expansion between 2nd and 4th dayof culture using tracking of cells expressing ER-mCherry marker, AtPIN5and AtPIN8. Error bars represent 95% confidence intervals.

FIG. 24. Quantitative analysis of PIN8 effect on auxin mediated cellexpansion. Concentration-depended cell expansion takes place fordeveloping protoplasts from wild type tobacco (a) and significant lossof auxin-mediated response in protoplasts isolated from AtPIN8-Venusoverexpressing line (b). At least 200 cells per group were analyzed.Error bars represent standard errors.

EXAMPLES

Some media and solutions used in the examples are listed in thefollowing tables:

TABLE 1 Solutions for protoplast isolation and immobilization. Unit ismM, pH 5.8-5.83 unless indicated otherwise.” PEG Alg-A W5^(c) TM550MMC600 MSC600 Alginic acid 2.8% (w/v) MES 10 mM 5 mM 10 mM 10 mMCaCl₂•2H₂0 125 mM 20 mM 20 mM Ca(NO₃)₂•4H₂0  67 mM Glucose 5 mM KCl 5 mMMgCl₂•6H₂0 10 mM 15 mM MgSO₄•7H₂O 10 mM Mannitol^(a) 270 mM 550 mOsm 550mOsm 600 mOsm NaCl 150 mM Sucrose^(b) 600 mOsm PEG1500 (g/l) 384.6^(a)approximately 90 g for 1 l of medium give 550 mOsm;^(b)approximately 165 g for 1 l of medium; ^(c)according to Menczel etal. Effect of radiation dosage efficiency of chloroplast transfer byprotoplast fusion in Nicotiana. Genetics 100, 487-495 (1982)

TABLE 2 Plant culture, preplasmolysis and culture protoplasts media.Unit is mg/l unless indicated otherwise, pH 5.8-5.83 F-PIN^(a) F-PCN^(b)SCN^(c) PCA^(d) SCA^(e) KNO₃ 1012 1012 2527.5 2527.5 2527.5 CaCl₂•2H₂O640 640 150 450 150 MgSO₄•7H₂O 370 370 246.5 746 1140 KH₂PO₄ 170 170NaH₂PO₄•H₂O 150 150 150 (NH₄)₂SO₄ 134 134 134 NH₄-succinate (mM) 20 20EDTAFe(III) Na Salt 40 40 40 40 40 KI 0.83 0.83 0.75 0.75 0.75 H₃BO₃ 6.26.2 3 3 3 MnSO₄•H₂O 22.3 22.3 10 10 10 ZnSO₄•7H₂O 8.6 8.6 2 2 2Na₂MoO₄•2H₂O 0.25 0.25 0.25 0.25 0.25 CuSO₄•5H₂O 0.025 0.025 0.025 0.0250.025 CoCl₂•6H₂O 0.025 0.025 0.025 0.025 0.025 Inositol 200 200 100 200100 Pyridoxine-HCl 2 2 1 2 1 Thiamin-HCl 1 1 10 1 10 Ca-panthotenate 2 22 Biotin 0.2 0.2 0.2 Nicotinic acid 2 2 1 2 1 MES 976 976 976 Sucrose130 20 20 15 Glucose 65 80 Coconut water (ml) 20 BAP 1 1 Dicamba 4 NAA0.1 0.1 0.5 Agar (g) 8 Gelrite (g) 2 ^(a,b,c)-according to Dovzhenko etal. (1998) Protoplasma 204, 114-118 ^(d,e)-according to Dovzhenko et al.(2003) Protoplasma 222, 107-111

TABLE 3 Artificial miRNAs (amiR) and their passenger strands (amiR*)amiR (5′-3′) SEQ ID NO: amiR* (5′-3′) SEQ ID NO: P1TAAGCGAATATATCTCAGCGC 136 GCACTGAGATATAATCGCTTT 137 P2TAAGCGAATATATCTCAGGGT 132 ACACTGAGATATAATCGCTTT 133 P3TAAATTACCATACATGCCTCT 128 AGCGGCATGTATGCTAATTTT 129 P4TTTGGGCGAAAACATCCCTCG 124 CGCGGGATGTTTTGGCCCAAT 125 P5TTCGAGTAAATATCGGACGTT 120 AAAGTCCGATATTAACTCGAT 121 P6TTCGAGTAAATATCAGACGTT 116 AAAGTCTGATATTAACTCGAT 117 P7TTTAAAACTAGAGCCACGCGG 112 CCACGTGGCTCTACTTTTAAT 113 P8TAAAGTTAGAGTTCCGACGAC 108 GTAGTCGGAACTCAAACTTTT 109 P9TGATTACGAATAAGTTTCCTG 104 CAAGAAACTTATTGGTAATCT 105 P10TAAGCGAATATATCTCGGCGC 100 GCACCGAGATATAATCGCTTT 101 P11TAACGTGGTAGAAGTGCGCGG 96 CCACGCACTTCTAGCACGTTT 97 P12TGATGCCGAATAAACTGGAGC 92 GCCCCAGTTTATTGGGCATCT 93 P13TTAGCCGTCATAACGTGGTGG 88 CCCCCACGTTATGTCGGCTAT 89 P14TTAGCCGTCATAACGTGGCAG 84 CTACCACGTTATGTCGGCTAT 85 P15TTAGCCGTCATAACGTGGTAC 80 GTCCCACGTTATGTCGGCTAT 81 P16TAAAGTTAGAGTTCCGACCGC 76 GCAGTCGGAACTCAAACTTTT 77 P17TATAATGGCAACATGGGGGGG 72 CCACCCCATGTTGGCATTATT 73 P18TATAATGGCAACATGCAGGGG 68 CCACTGCATGTTGGCATTATT 69 P19TAACGTGGTAGAAGTCCGCGG 64 CCACGGACTTCTAGCACGTTT 65 P20TAAAACTAGAGCCACGTGCCG 60 CGACACGTGGCTCAAGTTTTT 61 P21TTATAACGGAACCATAGCCCT 56 AGAGCTATGGTTCGGTTATAT 57 P22TTGATGCCGAATAAACTGCAG 52 CTACAGTTTATTCCGCATCAT 53 P23TGATTACGAATAAGTTTCCTC 48 GAAGAAACTTATTGGTAATCT 49 P24TCCAAAGTTAGAGTTGCGACG 44 CGCCGCAACTCTATCTTTGGT 45 P25TTATAACGGAACCATAGGCCT 40 AGACCTATGGTTCGGTTATAT 41 P26TATGATTAAAACTACAGCCGC 36 GCAGCTGTAGTTTAAATCATT 37 P27TATAATGAAACCTCCCAGGTC 32 GAACTGGGAGGTTACATTATT 33 P28TTTAAAACTAGAGCGACGCGG 30 CCACGTCGCTCTACTTTTAAT 31 P29TAAATTACCATACATGCCTTT 26 AACGGCATGTATGCTAATTTT 27 P30TATGACGGCAGGTCGAACGAG 22 CTAGTTCGACCTGGCGTCATT 23 P31TTTACCGAAACTAAACTGCTC 18 GAACAGTTTAGTTACGGTAAT 19 P32TTTGGGCGAAAACATCCCTGC 14 GCCGGGATGTTTTGGCCCAAT 15 P33TACGATTTGAACCATGAGGCC 10 GGACTCATGGTTCTAATCGTT 11 P34TAACGGTTTATGCCGCAGCGT 8 ACACTGCGGCATATACCGTTT 9 P35TGTTGGGCGAAAACATCCGTG 4 CAAGGATGTTTTCCCCCAACT 5 P36TAATATCAGACCTTGGAGCGT 138 ACACTCCAAGGTCAGATATTT 139 P37TCCAAAGTTAGAGTTCCGACG 134 CGCCGGAACTCTATCTTTGGT 135 P38TTTATGGGCAACGCGACCGAC 130 GTAGGTCGCGTTGGCCATAAT 131 P39TAACGGTTTATGCCCGAGCGT 126 ACACTCGGGCATATACCGTTT 127 P40TAATATCAGACCTTCAAGCGT 122 ACACTTGAAGGTCAGATATTT 123 P41TATGACTAGAGTGTTGCGGGG 118 CCACGCAACACTCAAGTCATT 119 P42TTAGTTGGAAGGTCTCGGACT 114 AGCCCGAGACCTTGCAACTAT 115 P43TTCGTTACTATTCCCCTGACG 110 CGCCAGGGGAATACTAACGAT 111 P44TTTATGGGCAACGCGGTCGAC 106 GTAGACCGCGTTGGCCATAAT 107 P45TATGACGGCAGGTCGAACGGC 102 GCAGTTCGACCTGGCGTCATT 103 P46TGAAGAGTTATGGGCAACGGG 98 CCAGTTGCCCATATCTCTTCT 99 P47TGTGGAGTAATCGGCGTGCTG 94 CAACACGCCGATTTCTCCACT 95 P48TATGACTAGAGTGTTCGGGGG 90 CCACCGAACACTCAAGTCATT 91 P49TGAAGAGTTATGGGCGACCCG 86 CGAGTCGCCCATATCTCTTCT 87 P50TAGATTCGAAGGTCTACGTCT 82 AGCCGTAGACCTTGGAATCTT 83 P51TAACGTGGTAGAAGTCCCGCG 78 CGAGGGACTTCTAGCACGTTT 79 P52TTCGAGTAAATATCAGGCCCT 74 AGAGCCTGATATTAACTCGAT 75 P53TGTTGGGCGAAAACGTCCGTG 70 CAAGGACGTTTTCCCCCAACT 71 P54TAAAGTTAGAGTTCGGACCGC 66 GCAGTCCGAACTCAAACTTTT 67 P55TAACGTGGTAGAAGTGCGCGG 62 CCACGCACTTCTAGCACGTTT 63 P56TGGAAAGAGAGGAGTGGGACG 58 CGCCCCACTCCTCACTTTCCT 59 P57TGGACGGCGAAGACGGCGACA 54 TGCCGCCGTCTTCCCCGTCCT 55 P58TGTCATCACACTTGTTGGCGG 50 CCACCAACAAGTGAGATGACT 51 P59TTGAAGTGGAAAGACAGGACT 46 AGCCCTGTCTTTCGACTTCAT 47 P60TTCCGGAGCATTGGTCGGGAG 42 CTACCGACCAATGGTCCGGAT 43 P61TACTGAACATAGCCATGCCTA 38 TAAGCATGGCTATCTTCAGTT 39 P62TTGAAGTGGAAAGAGACGACT 34 AGCCGTCTCTTTCGACTTCAT 35 GFP-6TTCTGGTAAAAGGACAGGGCC 28 GGACCTGTCCTTTAACCAGAT 29 GFP-7TTAATGATCAGCGAGTTGCAC 24 GTACAACTCGCTGTTCATTAT 25 GFP-9TTGTATTCCAACTTGTGGCCG 20 CGACCACAAGTTGCAATACAT 21 GFP-10TGATCAGCGAGTTGCACGCCG 16 CGACGTGCAACTCCCTGATCT 17 GFP-11TTGACTTCAGCACGTGTCTTG 12 CACGACACGTGCTCAAGTCAT 13 mockTATCATAAGAGCAGGTCCTGA 6 TCCGGACCTGCTCATATGATT 7

Example 1. Monolayer Embedding of Tobacco Leaf Protoplasts forVontinuous Cell Tracking

Wholly expanded leaves from 3-4 weeks old tobacco plant cultures(Nicotiana tabacum cv. Petite Havana) were used for protoplastisolation. Two leaves were cut in stripes 1-2 mm in width andpreplasmolysed for 1 h in 10 ml of F-PIN medium as described inDovzhenko et al. (1998) Protoplasma 204, 114-118. Preplasmolysis mediumwas replaced with 10 ml of fresh F-PIN supplemented with CellulaseOnozuka R-10 (DUCHEFA) and Macerozyme Onozuka R-10 (DUCHEFA) 0.25% each.Digestion was performed overnight (14 h) in the dark. Digestion mediumwas further filtered through 100 μm sieves in 12 ml tube (Greiner,Germany) to remove non-digested tissues. Afterwards 2 ml of TM550 wereoverlaid on a top of filtered F-PIN containing protoplasts. Intactprotoplasts were collected from the interlayer between TM550 andprotoplast/digestion mixture after 10 min flotation at 100 g andtransferred to a new tube. Total volume was adjusted to 10 ml with W5medium and protoplast number was estimated. Protoplasts were furtherpelleted for 5 min at 100 g and supernatant was discarded. After thelast centrifugation step protoplast pellet was mixed with TM550 toachieve density of 2×10⁴ cells per 1 ml. Protoplasts/TM550 mixture wasfurther mixed at ratio 1:1 with Alg-A medium for Ca²⁺-alginate embeddingor with TM550 containing 2% of low melting temperature agarose foragarose embedding. It is important to maintain temperature above 30° C.(recommended 35° C.) until formation of a cell layer at a well bottomand to avoid generation of agarose aggregates.

For cell immobilisation using low melting temperature agarose 100 μl or200 μl of protoplast embedding mixture per were transferred into 96-wellplates or 8-well slides respectively (FIG. 1A 1). After formation ofcell layers at 30-35° C. achieved either by centrifugation for at least1 min at 10 g or by sedimentation for at least 20-30 min (FIG. 1A. 2)plates can be placed at room temperature for agarose solidification(FIG. 1A 3). Embedded cells should be washed twice for 15 min with theculture F-PCN medium and 200 μl of fresh F-PCN were added after the lastwashing (FIG. 1A 4). It is important to note, that within first 24 h ofculture mild expansion of agarose gel with embedded cells was notavoidable due to long lasting water uptake from the culture medium.

For cell immobilisation using Ca²⁺-alginate embedding 100 μl or 200 μlof protoplast embedding mixture per were transferred into 96-well platesor 8-well slides respectively (FIG. 1B 1). To form a cell layerprotoplasts could be either centrifuged at 10 g for at least 1 min orsedimented for at least 20-30 min (FIG. 1B 2). Afterwards the uppersurface of the protoplast embedding mixture was covered with 20 μl or 40μl for 96-well plates or 8-well slides respectively of W5 or anotherosmotically adjusted solution with a high Ca²⁺-salt 20 mM) content in aform of micro-droplets 1 μl droplets if manually or 100-500 nl dropletsif using dispersing robots, FIG. 1B 3, FIG. 2). After 5 min final volumeof W5 medium was adjusted to 200 μl and after additional at least 15 mincell immobilisation was achieved (FIG. 1B 4). W5 medium was replacedwith 200 μl of the culture F-PCN medium for washing. After two washings,200 μl of fresh F-PCN were added for subsequent protoplast culture (FIG.1B 5). Embedded cells were observed with 24 h interval and theirdevelopment was documented by imaging of the same area using invertedmicroscope (Axiovert 200M, Zeiss) for a period of 1 week (FIG. 3). Dueto formation of a single cell layer at the bottom of culture wells andsubsequent immobilization this technique was namedprotoplast-monolayer-embedding technique (PME).

Example 2. Effect of R113 Compound on Development of Tobacco LeafProtoplasts

Protoplast isolation was done as described in the example 1 prior thewashing with the culture medium. Cell immobilisation was performed in8-well slides (IBIDI). After the removal of W5 medium, F-PCN withvarious concentration of compound R113 per well was used for washing andfurther cell culture. Analysis of cell division efficiency (FIG. 4) wasperformed using inverted microscope (Axiovert 200M, Zeiss).

Example 3. Monolayer Embedding of Arabidopsis Protoplasts for ContinuousCell Tracking

Arabidopsis protoplasts were isolated from hypocotyls of 7-days oldArabidopsis seedlings (Col-0) germinated on SCA medium according toDovzhenko et al. (2003) Protoplasma 222, 107-111. Explants were cut in0.5-1 mm fragments and preplasmolysed in MMC600 for 1 h. Afterwards themedium was substituted with fresh MMC600 supplemented with 0.5%Cellulase Onozuka R-10 (DUCHEFA), 0.5% Macerozyme Onozuka R-10 and 0.05%Driselase (SIGMA). After 14 h of digestion, protoplast containing mediumwas filtered through 32 μm sieves. Protoplasts containing medium wasfurther mixed with an equal volume of TM550 and collected bycentrifugation for 10 min at 100 g. Supernatant was removed and thepellet was mixed with TM550:Alg-A mixture (1:1) to achieve density 1·10⁴protoplasts per 100 μl. 100 μl aliquots of protoplast embedding mixturewere transferred in 96-well plate and immobilized as described in theexample 1. After the removal of W5 medium, PCA medium was used forwashing and culture steps. Cell observations (FIG. 5) were done usinginverted microscope (Axiovert 200M, Zeiss).

Example 4. Analysis of Promoter Activity Using Monolayer Embedding

Cotyledons from 7-days old Arabidopsis seedlings (DR5-GFP line, Col-0background) were removed and cut in 0.5-1 mm fragments. Preplasmolysisand digestion was performed as describe in the example 3, however noDriselase was used for digestion. After filtration through 56 μm sieves,protoplasts were collected in 12 ml tube and pelleted for 10 min at 100g. Supernatant was discarded and 10 ml of MSC600 was added. Forflotation 2 ml of TM550 were overlayed on a top of MSC600 andcentrifuged for 10 min at 100 g. Interlayer was collected andtransferred into a new 12 ml tube. Total volume was adjusted to 10 mlwith W5 medium. Protoplasts were washed for 5 min at 50 g and afterwardspellet was resuspended in TM550 to achieve density 2·10⁴ protoplasts per100 μl. After mixing 1:1 with Alg-A medium, 100 μl aliquots ofprotoplast embedding mixture were used for cell immobilisation in96-well plates. Immobilisation and subsequent culture procedure wereperformed as described in example 3. While GFP fluorescence was detectedonly in 3% of freshly isolated protoplasts, it was observed in over than95% of intact cells after 48 h of culture thus demonstrating activity ofDR5 promoter (FIG. 6).

Example 5. Transient Transformation of Arabidopsis Shoot ProtoplastsUsing Dried DNA

Purified plasmid DNA was dissolved in ultra-pure sterile water. Aliquotsof aqueous DNA solution at various concentrations (0, 0.1, 0.5, 1 and 5μg per 10 μl) were transferred inside 96-well plate (ABIGene). Waterevaporation took place under the sterile bench overnight (FIG. 1C 1,2).After complete evaporation plates could be used either immediately orstored at −20° C.

Arabidopsis shoots from 3-weeks old seedlings (Col-0) were used.Digestion and isolation were performed as described in example 4. Afterlast washing step cell density was adjusted to 1×10⁵ and aliquots of 30μl were transferred into the wells containing dried DNA (FIG. 1C 3).Afterwards cells were left to sediment for at least 2 min (FIG. 1C 4).Alternatively, protoplasts could be centrifuged for 1 min at 10 g.Further PEG-mediated DNA uptake was performed by adding an equal volumeof 40% PEG1500 solution to protoplasts in wells (FIG. 1C 5). After 8 minof the PEG treatment, a half of the total volume of TM550 was added andafter additional 2 min total volume was increased to 1 ml using TM550(FIG. 1C 6). To avoid formation of protoplast-aggregates, well contentswere mixed using 1 ml 8-well pipette. Sedimentation for at least 30 minwas performed to collect cells. Alternatively, protoplasts could becentrifuged for 10 min at 50 g. Supernatant (950 μl) was discarded andprotoplast pellet was resuspended in 300 μl of PCA. Transformationefficiencies (FIG. 7) were estimated after 30 hours using invertedmicroscope (Axiovert 200M, Zeiss).

Co-transformation with at least two (or more) either plasmids or PCRamplified fragments was performed. Plasmids carrying expression cassetteof GFP or endoplasmic reticulum-mCherry were either mixed and dried uponwater evaporation under a sterile bench inside 96-well plates or mixedand used directly for transformation. Transformation was carried out asdescribed above. Equal amount of each plasmid was used (0.5 μg perplasmid). To simulate a simple pipetting robot, all manipulations(adding cells, adding reagents prior washing step after transformation)were performed by using multichannel pipette. Cells were added directlyto wells containing either dried DNA or DNA dissolved in water and nofurther mixing was performed prior adding PEG1500 solution. Comparisonof transformation and co-transformation efficiencies was performed after24 h. Images were acquired using iMIC (TILL Photonics, Germany)automated microscope, and image analysis was performed using ImageJ freesoftware. At least 250 cells per individual transformation were analysed(Table 4).

TABLE 4 Analysis of GFP and ER-mCherry co-transformation efficienciesusing dried DNA or DNA dissolved in water. Each line represents onetransformation experiment. Co-transformed cells Co-transformed out ofall transformed, % cells out of all cells, % Dried DNA in Dried DNA inDNA solution DNA solution 92.41 88.0 20.33 6.1 85.15 86.0 12.76* 5.994.12 85.7 21.05 7.2 92.31 91.6 20.47 13.9 91.39 88.4 19.11 13.4 92.4184.6 18.31 17.6 96.2 34.2 93.5 42.1 87.7 31.2 *wrong amount of PEG (~½of required volume) was added during this transformation, due to defectof pipette's channel

Significant variation of co-transformation efficiencies was observedusing DNA dissolved in water, while using dried DNA approach highlyreproducible results could be obtained (FIG. 8). This criteria isessential for automation of the whole procedure.

Example 6. Transient Transformation of Tobacco Leaf Protoplasts UsingDried DNA in Combination with Continuous Cell Tracking

Plasmid DNA aliquots (1 μg per 10 μl of ultrapure water) were dried asdescribed in the example 5 (FIG. 1C).

Tobacco leaf protoplasts were isolated as described in the Example 1prior the embedding procedure. After protoplast pelleting in W5 mediumand removal of the supernatant, cell density was adjusted to 2·10⁶cells/ml with TM550. Aliquots of 30 μl (2-2.5·10⁵ cells) weretransferred into the wells using 8-channel pipette. Afterwards cellswere left to sediment for at least 2 min. Alternatively, protoplastscould be centrifuged for 1 min at 10 g. Further PEG-mediated DNA uptakewas performed by adding an equal volume of 40% PEG1500 solution toprotoplasts in wells. After 8 min of the PEG treatment, a half of thetotal volume of TM550 was added and after additional 2 min total volumewas increased to 1 ml using TM550. To avoid formation ofprotoplast-aggregates, well contents were mixed using 1 ml-tip pipette.Sedimentation for at least 30 min was performed to collect cells.Alternatively, protoplasts could be centrifuged for 10 min at 50 g.Supernatant (950 μl) was discarded and protoplast pellet was resuspendedin 250 μl of TM550. Protoplasts were further mixed with Alg-A medium forembedding. Embedding was further performed as described in the example 1(FIG. 1B). After the embedding transformation efficiencies, proteinlocalisation and effect of gene expression was analysed using automatedinverted microscope (iMIC or MORE, TILL Photonics, FIG. 9) and confocallaser scanning microscope (LSM5META, Zeiss, FIG. 10).

Example 7. RNAi-Mediated Gene Knock-Down Using Artificial miRNAs

Arabidopsis shoots from 3-weeks old seedlings (35S::GFP in Col-0background) were used. Digestion and isolation were performed asdescribed in example 4. After last washing step cell density wasadjusted to 1·10⁵ and aliquots of 30 μl were transferred into 96-wellplate. 1-2 μg DNA in 5 μl of ultra-pure sterile water was mixed withprotoplasts. PEG-mediated DNA uptake was performed by adding an equal(35 μl) volume of 40% PEG1500 solution. After 8 min of the PEGtreatment, a half of the total volume of TM550 was added and afteradditional 2 min total volume was increased to 1 ml using TM550. Aftercareful mixing with 1 ml pipette for separation of protoplastaggregates, sedimentation for at least 30 min was performed to collectcells. Supernatant (950 μl) was discarded and protoplast pellet wasresuspended in 75 μl of TM550. Protoplasts were further mixed with Alg-Amedium for embedding. Embedding and subsequent culture were performed asdescribed in the example 3. Effect of artificial miRNA (amiRNA) on geneexpression (FIG. 11) was analysed for a period of 6 days using automatedinverted microscope (Axiovert 200M, Zeiss).

Example 8. Screening of Artificial miRNA Efficiency

Arabidopsis shoots from 3-weeks old seedlings (Col-0) were used.Digestion, isolation, and culture procedures were performed as describedin example 5. Transformation was performed as described in the example7. Efficiencies of amiRNAs were estimated as a read-out of fluorescenceintensity of the target gene translationally fused with the reportergene (FIG. 12). Randomly selected amiRNA constructs were further usedfor generation of transgenic plants which exhibited phenotypesconfirming microscopy-based conclusions on amiRNA efficiency (FIG. 13).

Example 9. Cloning Free Screening Approach in Plant Cells

Experiment 1. Transient Transformation and Co-Transformation Using PCRProduct Transient Transformation (PPTT)

We compared impact of extra flanks around the“promoter-gene-GFP-terminator” expression cassette againsttransformation using plasmid DNA. Protoplast isolation andtransformation using either tobacco or Arabidopsis leaf protoplasts wasperformed as previously described in this application. PCR products wereamplified as shown on FIG. 17. Primers were designed to match thisdesign: P1-gactagagccaagctgatctcctt (SEQ ID NO:140),P2-aggtcactggattttggttttagg (SEQ ID NO:141), P3-tgccggtgatcttctcggaaaaca(SEQ ID NO:142), P4-agaaaccatcggcgcagctattta (SEQ ID NO:143),P5-tcacttcctcgctgcgctcaagtg (SEQ ID NO:144), P6-tcgtattgggaatccccgaacatc(SEQ ID NO:145).

Transformation using dried or liquid PCR-amplified DNA product wasperformed, and both transformation procedures were efficient.Intriguingly, PPTT is working much more efficient in comparison to astandard, plasmid DNA-based transient transformation (FIG. 18).Furthermore, for plant species such as tobacco, in which transformationefficiencies when using plasmid DNA are always worse than in Arabidopsisincreased number of transformed cells was observed (FIG. 18).

Experiment 2. Co-Expression of ER-mCherry Marker and At1g01730-GFP inTobacco and Arabidopsis Protoplasts.

After PCR amplification and purification, ER-mCherry and At1g01730-GFPexpression cassettes were mixed, dried, and PEG-mediated DNA uptake wasperformed. Microscopy analysis revealed co-expression of both genes inmore than 95% of transformed cells for both, Arabidopsis and tobaccoprotoplasts.

Experiment 3. amiRNA, Split-FP Screenings Using PPTT

Our further aim to combine PPTT with amiRNA and other functionalscreenings such as split-GFP, split-YFP etc. Since co-expression ofvectors works perfect, split screenings could be done straight awayusing confirmed interacting pair. Transformation with the luciferasereporter and subsequent luciferase activity measurements weresuccessfully tested using auxin sensor construst (FIG. 19).

The amiRNA fluorescence based marker screening can be redesigned withluciferase activity evaluation using PPTT (FIG. 20). This design willallow completely automated sample manipulations, measurements and datareading where human will be responsible only for providing consumablesand material, and transfer the screening plates from the robot toincubator and a reading instrument (microscope, plate-readers etc.). Ifneeded, each step can be automated.

Example 10. Quantitative Analysis of Cellular Expansion Using PIN8, anIntracellular Modulator of Auxin Homeostasis

Plant hormone auxin is an important regulator of plan growth,development and responses to environmental stimuli. The molecularmechanisms to actively transport this compound was evolved achieving themaximal complexity in higher plants. There are several protein familieswhich are involved in regulation of auxin transport and generation ofauxin gradients. One of them is PIN protein family which consists of 8members in Arabidopsis, AtPIN1-AtPIN8. AtPINs are expressed specificallyin different tissues being typically localized to the plasma membrane.Understanding of AtPIN function was crucial to understand the mechanismof auxin efflux as well as the auxin gradient formation in tissues andorgans. However, currently there is no understanding, of how auxin ismoved within the cell until it reaches the nucleus, where auxin-mediatedregulation of gene expression takes place. Here we used single plantcells and intracellularly localized AtPIN5 and AtPIN8 to address thisquestion.

Experiment Design:

We compared the effect of auxin concentration on cell elongation over acontinuous period (4-6 days) using tobacco leaf protoplasts from wildtype tobacco and from the tobacco line overexpressing AtPIN8translationally fused with Venus fluorescent marker protein(PIN8VenusOx). Two strategies were used:

-   -   effect of PIN5 and PIN8 expression on cell elongation using        transient transformation with dried DNA, protoplast        immobilization and culture with manual cell imaging and        tracking.    -   effect of auxin concentration (0, 1 pM, 10 pM, 100 pM, 500 pM, 1        nM, 10 nM, 100 nM, 500 nM, 1 μM, 2.5 μM, 5 μM, 10 μM, 25 μM and        50 μM) and quantitative analysis of cell elongation to compare        wild type and PIN8VenusOx protoplasts using automated imaging        (every 24 h for 4 days, 5 reference points) and computational        image analysis.

Experiment 1. Functional Analysis of AtPIN5, AtPIN8 and AtPIN8VenusExpression in Tobacco Leaf Arotoplast.

Three plasmids were generated and used:

-   -   pAM-PAT-mCherry-PIN5 (PIN5, FIG. 21)    -   pAM-PAT-mCherry-PIN8 (PIN8, FIG. 21)    -   p2GW7,0-PIN8Venus (PIN8-Venus, FIG. 21)

pAM-PAT-mCherry-PIN5 and pAM-PAT-mCherry-PIN8 (both have backbone as inpAM-PAT-AtPIN1, FIG. 16 of the current application) contain mCherryexpression cassette to identify transformed cells (transformationmarker) and PIN5 or PIN8 expression cassettes without tags respectively.These constructs were used to compare the effect of PIN5 and PIN8expression during protoplast development using cell tracking. Effect ofVenus tag insertion in the coding sequence of AtPIN8 was compared usingpAM-PAT-mCherry-PIN8 and p2GW7,0-PIN8Venus (this plasmid was used alsoin the Example 6, and FIG. 15 of the current application). Asillustrated on images below, we observed no effect of PIN5 on cellelongation, while both, AtPIN8 and AtPIN8Ox had a similar effectresulting in enhanced cell elongation.

Transient PIN5 expression does not affect developmental program oftobacco leaf protoplasts, while expression of PIN8 arrests cell divisionand enhances elongation (FIG. 21).

Experiment 2. Quantitative Analysis of Transient AtPIN8 Expression onExpansion of Tobacco Leaf Protoplasts.

ER-targeting of AtPIN5 has been previously reported (Mravec et al.(2009) Subcellular homeostasis of phytohormone auxin is mediated by theER-localized PIN5 transporter. Nature 459:1136-1140) we usedSpinning-disk and TIRF microscopy method to analyze localization ofAtPIN8, which is also targeted to ER (FIG. 22). It is important tostress, that our immobilization method allows using TIRF approach, whichallows to study processes near plasma membrane and is possible only ifthe object (cell) is in a close proximity to a surface of imagingslide/plate.

Therefore quantitative analysis and comparison of ER-marker, PIN5 andPIN8 was performed. pFGC19-ER-yb (Nelson B K et al. (2007) A multi-colorset of in vivo organelle markers for colocalization studies inArabidopsis and other plants Plant Journal 51:1126-1136) andpAM-PAT-mCherry-PIN5 and pAM-PAT-mCherry-PIN8 were used to transientlytransform tobacco leaf protoplasts. Cell tracking, cell divisionanalysis and quantitative measurements of cell diameter using ImageJfreeware were performed manually. AtPIN5 did not affect cell divisionrates in analyzed cells, while PIN8 inhibited cell divisions (Table 1).Only AtPIN8 enhanced cell elongation (FIG. 23).

TABLE 5 Analysis of cell division rates in transiently transformedcells. Sample ER PIN5 PIN8 total number of cells 26 26 30 dead cells(6dat) 3 6 12 non-dividing cells (6dat) 10 7 18 divided cells (6dat) 1313 0 dead cells (6dat), % 11.5 23 40 non-divided cells (6dat), % 38.5 2760 divided cells (6dat), % 50 50 0

Experiment 3. Modulation of Intracellular Auxin Homeostasis Using AtPIN8and Quantitative Analysis

Protoplasts were isolated and immobilized using our procedure describedin the current patent application. Culture medium with final auxinconcentration of 0, 1 pM, 10 pM, 100 pM, 500 pM, 1 nM, 10 nM, 100 nM,500 nM, 1 μM, 2.5 μM, 5 μM, 10 μM, 25 μM and 50 μM was used for washingsteps and cell culture afterwards in corresponding wells. Automatedimage acquisition in a form of volume stacks was performed using iMICmicroscope (TILL Photonics GmbH, Germany) in 24 h interval starting fromthe embedding (0 h, 24 h, 48 h, 72 h, 96 h). Recording coordinates werestored, and the plate was removed from the microscope after each imagingsession. This results in non-significant shift in imaging areas, whichdid not affect further image analysis, and these cannot be achievedusing any other existing protoplast culture systems at this or evengreater scale. Image analysis (segmentation, cell identification, cellclassification and parameter measurements, in this particular case acell diameter) were performed by a tool which was specificallydeveloped. This experiment allowed to extract quantitative informationfor hundreds cells per group, the whole experiment was performed in asingle 96-well plate. Several training loops using expert knowledge wereperformed to achieve accuracy of cell identification over every timepoint (above 90%). This data were used for analysis of PIN8 effect (FIG.24) and currently provide the basis of the primary mathematical modeldescribing how auxin should move within the cell in order to achieve thenucleus and regulate gene expression.

The invention claimed is:
 1. A method for introducing a polynucleotideinto non-adhesively growing plant cells, comprising the following steps:a. providing a solid support having a polynucleotide in a dry stateimmobilized thereto; b. contacting non-adhesively growing plant cellswith the polynucleotide on the solid support of step (a) so as to obtaintransformed non-adhesively growing plant cells, wherein step (b)comprises: i. adding a suspension comprising the non-adhesively growingplant cells to the solid support of step (a), ii. sedimenting thenon-adhesively growing plant cells, so as to arrange them in a layer onthe solid support of step (a), iii. adding a transformation agent to thesuspension; and iv. optionally removing the transformation agent fromthe non-adhesively growing plant cells; and c. optionally washing thenon-adhesively growing plant cells.
 2. The method of claim 1, whereinstep (a) comprises adding a solution containing the polynucleotide ontothe solid support and removing the water from the solution on the solidsupport.
 3. The method of claim 1, wherein after step (iii) thenon-adhesively growing plant cells are incubated for 1 to 30 minutes inthe presence of the transformation agent so as to obtain the transformednon-adhesively growing plant cells and/or said transformation agent isselected from the group consisting of polyethylene glycol (PEG),poly-L-ornithine, polyvinyl alcohol and divalent ions.
 4. The methodaccording to claim 1, wherein, in step (a), at least 3 differentpolynucleotides are immobilized on the same solid support, eachpolynucleotide being spatially separated from the other polynucleotidesand/or the solid support has a plurality of locations and eachpolynucleotide is immobilized at a separate location.
 5. The method ofclaim 1, wherein said non-adhesively growing plant cells are plantprotoplast cells.
 6. The method of claim 1, wherein said polynucleotidecomprises (i) a nucleic acid sequence encoding an artificial microRNA,(ii) a nucleic acid sequence representing a target gene of theartificial microRNA, and (iii) optionally a nucleic acid sequenceencoding a transformation marker.
 7. A method for analyzingnon-adhesively growing cells, comprising the following steps: a.introducing a polynucleotide comprising at least one coding sequenceinto non-adhesively growing cells by a method according to claim 1 toobtain transformed non-adhesively growing cells; b. culturing thetransformed non-adhesively growing cells under conditions that allowexpression of the at least one coding sequence comprised in thepolynucleotide; c. arranging the transformed non-adhesively growingcells in a monolayer and immobilizing them in the monolayer; and d.detecting at least one parameter by microscopic analysis.
 8. The methodof claim 7, wherein (i) the immobilization of the non-adhesively growingcells in a monolayer is achieved by adding a gelling substance to thenon-adhesively growing cells, centrifuging the protoplast cells toobtain a monolayer of non-adhesively growing cells, and solidifying thegelling substance to form a gel in which the non-adhesively growingcells are embedded and/or (ii) said at least one parameter is selectedfrom the group consisting of fluorescence, luminescence, morphology andcombinations thereof.
 9. A screening method to identify efficient plantmicroRNA sequences, comprising the following steps: a. introducing apolynucleotide into non-adhesively growing plant cells by the method ofclaim 6 so as to obtain transformed non-adhesively growing plant cells;b. culturing the transformed non-adhesively growing plant cells underconditions that allow expression of at least the nucleic acid sequenceencoding the artificial microRNA, and the nucleic acid sequencerepresenting the target gene of the artificial microRNA; c. designatingthe artificial microRNA sequence that is capable of inhibitingexpression of the target gene as an efficient plant microRNA sequence.10. The screening method of claim 9, wherein (i) the target gene islabeled with a first fluorescent protein, and the transformation markeris labeled with a second fluorescent protein and/or (ii) at least 24different artificial microRNAs are examined in one screening cycle usingone single solid support wherein the inhibition of expression of thetarget gene is determined by microscopy and/or (iii) the inhibition ofexpression of the target gene is determined by microscopy.
 11. Themethod of claim 1, wherein said polynucleotide is a lineardouble-stranded DNA consisting of a promoter, an open reading frame, anda terminator, and wherein said nucleic acid is directly used for thetransformation without inserting it into a plasmid.
 12. The methodaccording to claim 1, wherein said solid support has a plurality ofseparate cavities, each of which has said polynucleotide immobilized atits bottom.
 13. The method of claim 1, wherein said non-adhesivelygrowing plant cells are selected from the group consisting of cells fromseeds, suspension cultures, embryos, meristematic regions, callustissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, andmicrospores.
 14. The method of claim 1, wherein said non-adhesivelygrowing plant cells comprise monocotyledonous or dicotyledonous plantcells.
 15. The method of claim 1, wherein said non-adhesively growingplant cells comprise algae or mosses such as Physcomitrella patens. 16.The method of claim 1, wherein said non-adhesively growing plant cellsare microspores.
 17. An automated method for analyzing a cell, saidmethod comprising the following steps: a. providing a solid supporthaving a polynucleotide in a dry state immobilized thereto; b. adding aculture of non-adhesively growing cells to the solid support; c.arranging the non-adhesively growing cells in a monolayer on the solidsupport and immobilizing said cells in said monolayer; d. adding atransformation agent to the culture so as to obtain transformednon-adhesively growing cells; e. optionally removing the transformationagent from said transformed non-adhesively growing cells; f. optionallywashing said transformed non-adhesively growing cells; and g. detectingat least one parameter of said transformed non-adhesively growing cellsby microscopic analysis.
 18. The method of claim 17, wherein (i) saidnon-adhesively growing cells are plant protoplast cells, and/or (ii) theimmobilization of the non-adhesively growing cells in a monolayer isachieved by adding a gelling substance to the non-adhesively growingcells, centrifuging the protoplast cells to obtain a monolayer ofnon-adhesively growing cells, and solidifying the gelling substance toform a gel in which the non-adhesively growing cells are embedded,and/or (iii) said at least one parameter is selected from the groupconsisting of fluorescence, luminescence, morphology and combinationsthereof.
 19. The method of claim 17, wherein said polynucleotidecomprises a linear double-stranded DNA consisting of a promoter, an openreading frame, and a terminator, and is directly used for thetransformation without inserting it into a plasmid.